Discharge of industrial waste water containing inorganic cyanide into rivers, lakes and sea water causes serious environmental pollution. Hereinafter inorganic cyanide comprises cyanide ions and hydrogen cyanide.
Cyanide containing waste water is produced by a great variety of industrial manufacturing processes such as mining of precious metals, electroplating and production of synthetic fibres, polymers, food additives and food products.
Due to its high toxicity and abundant use in industrial processes, inorganic cyanide is among the highest ranking pollutants on EPA's List of Priority Pollutants (cf. Environ.Sci.Technol. 13 (1979), 416-422).
Some European countries have issued regulations demanding that waste water discharged into recipient waters must contain less than about 0.5 ppm of inorganic cyanide.
It is well established that about 2,000 species of higher plants are cyanogenic (i.e. contain organic cyanide compounds) and that these plants release significant amounts of inorganic cyanide upon decay or during post harvest processing. Cyanogenic plants include important agricultural crops such as cassava, sorghum, alfalfa, beans, peaches and almonds, some of which may contain high levels of cyanide (up to about 3 g of cyanide per kg of plant tissue).
In areas where the above-mentioned crops constitute the main diets, whole populations are likely to suffer from chronic cyanide poisoning (cf. page 101 and 122 in Cyanide in Biology, B. Vennesland, E. E. Conn, C. J. Knowles, J. Westley and F. Wissing, Eds., 1981, Academic Press, London and New York).
Degradation, conversion and detoxification of inorganic cyanide is, therefore, a matter of great importance in connection with 1) waste water treatment, 2) chemical manufacturing and 3) human nutrition.
Pure chemical processes for the conversion of the inorganic cyanide in cyanide containing waste streams have been practiced for many years. Conventional chemical treatment methods include alkaline chlorination, hydrogen peroxide treatment, ion exchange or electrolysis. These processes are expensive and in many instances impractical on a large scale. Furthermore, alkaline chlorination is a destructive process which may lead to the formation of chlorinated organic compounds which themselves are serious pollutants.
Several biological methods of treating cyanide containing waste waters have been proposed and practiced including acclimated sludge processes and various biological filter systems (cf. Howe, R.H.L., 1965, Int.J.Air Water Poll. 9, 463-478).
U.S. patent specifications Nos. 3,756,947, 3,940,332, 4,461,834 and 3,660,278 teaches the use of specially prepared sludge systems comprising 1) the seeding of activated sludge with cyanide converting microorganisms and 2) acclimation of the mixed system for one to several weeks prior to use. The sludge based systems described in these four U.S. patent specifications have, according to their claims, a very limited capacity of cyanide conversion--only waste water containing less than 50-250 ppm (2-10 mM) of cyanide ion can be efficiently decontaminated. Industrial waste water may contain considerably higher concentrations of cyanide.
Biotreatment systems using any kind of adjusted sludge with or without the addition of specific and viable cyanide converting microorganisms may be difficult to maintain and are not suitable for small scale operation. Also, sludge based systems are restricted to waste water treatment and obviously not practicable for, for example, cyanide conversion in food processing and for removal of cyanide residues in chemical product streams.
Enzymatic processes for the conversion of cyanide to formamide using fungal cells or immobilized fungal cells have been described in two European patent (publication Nos. 61,249 and 116,423). These patents claim the use of enzymes (enzyme: formamide hydro-lyase alias cyanide hydratase, EC 4.2.1.66) all of which were produced from fungi, mostly species of known phytopathogenic fungi. Cyanide was converted with 98% efficiency or down to about 1 ppm of inorganic cyanide at a temperature of 0-35.degree. C. and a pH value of 6-10. Widespread technical use of cyanide converting enzymes produced from phytopathogenic fungi is however, problematic unless the commercial products are made completely sterile with respect to production strains.
There have been several reports (cf. Knowles and Bunch (Adv.Micr.Physiol. (1986), 73-111, Eds.: A. H. Rose and D. W. Tempest, Academic Press, London and New York) on the isolation of cyanide converting bacteria. Some of these bacterial strains are no longer available and according to the reports none of these bacteria produce enzymes which can convert high concentrations of inorganic cyanide to very low concentrations.
In none of these reports the enzymatic pathways of the cyanide conversions and the intermediates of the reactions have been identified clearly.
For example, bacterial isolates ATCC 39204 (U.S. patent specification No. 4,461,834); ATCC 21697 and ATCC 21698 (U.S. patent specification No. 3,756,947); ATCC 21930 (U.S. patent specification No. 3,940,332) and ATCC 21419 (U.S. patent specification No. 3,660,278) were claimed to have cyanide converting properties. Strains ATCC 21697 and ATCC 21698 stated in U.S. patent specification No. 3,756,947 to be Achromobacter nitriloclastes and Alcaligenes viscolactis, were reclassified as Bacillus subtilis and Corynebacterium sp., respectively, upon deposition at ATCC (ATCC: American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, USA). Of the last-mentioned 5 strains thus quoted as producers of cyanide converting enzymes, only ATCC 21930 (Corynebacterium sp.) was actually found to exhibit such activity when tested thoroughly in our laboratory. The cyanide converting enzyme system of ATCC 21930 was, however, found to be inactivated rapidly at low cyanide concentrations (less than or equal to 20 mM). At higher concentrations, the enzyme system was found to be completely inactive.
The scientific information available through patents cited hereinbefore and articles cited in the above recent review paper by Knowles and Bunch indicate that factors such as phytopathogenicity of production strains, enzyme inhibition and enzyme inactivation by cyanide are the primary obstacles to the technical use of enzymes for cyanide detoxification.
It has now surprisingly been found that certain gram-negative bacterial isolates belonging to the genus Alcaligenes produce cyanide converting enzymes with properties which are highly superior to the ones described to date. Such superior properties include e.g., tolerance towards high cyanide concentrations and substrate kinetic features which allow detoxification of cyanide to very low concentrations. Two bacterial isolates producing such superior cyanide converting enzymes have both been identified by DSM (Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany) as species of Alcaligenes denitrificans subs. denitrificans and they have been deposited at DSM under the numbers DSM 4009 and DSM 4010 in connection with the present patent specification. The cyanide converting enzymes of DSM 4009 and DSM 4010 which catalyze the direct hydrolysis of inorganic cyanide to formic acid and ammonia are clearly different from other known cyanide converting enzymes such as: cyanide hydratase (formamide hydro-lyase E.C. 4.2.1.66), cyanide oxygenase, B-cyanoalanine synthase and others described in the paper by Knowles and Bunch (in Adv.Micr.Physiol. (1986), 73-111, cf. above).
The name Cyanidase is, therefore, proposed as a new name for this novel enzyme type produced by DSM 4009 and DSM 4010; an enzyme class which catalyzes the direct conversion of inorganic cyanide to formic acid and ammonia (i.e. without the intermediacy of formamide).
Cyanide converting enzymes (including cyanidase) have not previously been described in other Alcaligenes species and test in our laboratory has shown that they are not produced either constitutively nor inducibly by registered type strains of Alcaligenes denitrificans subs. denitrificans (DSM 30026 and NCIB 11961 (National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland) nor by Alcaligenes species deposited as NCIB 10109, NClB 8687, ATCC 31371 and DSM 30030.
It, therefore, seems appropriate to recognize the two isolates DSM 4009 and DSM 4010 of this invention either a new species of Alcaligenes or as new subtypes (variants) of Alcaligenes denitrificans characterized by their elaboration of a novel type of cyanide hydrolyzing enzyme--a so-called Cyanidase.
The enzymes of the present invention which are capable of converting inorganic cyanide are consequently hereinafter designated "cyanidases" or "cyanide converting enzymes".